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Original Article

Botanical Medicines With Activity Against Stationary Phase Bartonella henselae

Ma, Xiao1; Leone, Jacob2; Schweig, Sunjya3; Zhang, Ying4

Editor(s): van der Veen, Stijn

Author Information
Infectious Microbes & Diseases: September 2021 - Volume 3 - Issue 3 - p 158-167
doi: 10.1097/IM9.0000000000000069

Abstract

Introduction

Bartonella species are fastidious Gram-negative facultative intracellular pathogens with a unique intraerythrocytic lifestyle, which usually require an obligately bloodsucking arthropod vector and a mammalian host during their life cycle.1,2 At least thirteen Bartonella species are known to be pathogenic in humans, leading to either acute or chronic infections with known diseases such as cat scratch disease, trench fever, Carrion disease, and bacillary angiomatosis.3 Infection typically begins with cutaneous inoculation, followed by the bacteria subsequently establishing a primary niche in the endothelium1 before moving into the bloodstream and later intraerythrocytic infection.2

Bartonella henselae is the causative agent of cat scratch disease, which can manifest symptoms including fever, lymphadenopathy, malaise, abdominal pain, and arthritis that can persist for several months.4 Disseminated B. henselae may lead to the development of serious complications such as ocular manifestations, including neuroretinitis,5 neurologic manifestations, including seizures, cranial nerve palsies, and aseptic meningitis,4 visceral organ involvement, including hepatomegaly and/or splenomegaly,4 and cardiac manifestations, including endocarditis.3 Case studies have implicated B. henselae in monoclonal and biclonal gammopathy6 and various autoimmune manifestations, including pediatric acute-onset neuropsychiatric syndrome,7 transverse myelitis,3 and autoimmune thyroiditis.8Bartonella has been described as a “stealth pathogen”9 due to its ability to cause persistent bacteremia, evade the immune system, and cause varying types and severity of symptomatology.9,10Bartonella can induce vasoproliferative tumor formation11 and a recent review discussed the need to research Bartonella's potential role in breast cancer tumorigenesis.12

Emerging evidence suggests B. henselae may also serve as a co-infective pathogen in patients with other vector-borne diseases.12 In particular, ticks are known to be polymicrobially infected with multiple pathogens,13 including Borrelia burgdorferi, the most common etiologic agent in Lyme disease. Although the ability of ticks to serve as a competent vector for transmitting B. henselae to humans is debated, ticks have been documented to carry B. henselae12 and case reports have linked B. henselae-related diseases to a tick attachment.14,15 Multiple papers have documented B. henselae exposure in patients with Lyme disease and patients with co-infections may experience more severe and protracted clinical manifestations.15–18

Currently, there is no single treatment effective for Bartonella-associated diseases and antibiotic recommendations differ depending on specific presentations.19 The first-line antibiotics for treatment of Bartonella-associated infections include doxycycline (DOX), erythromycin, azithromycin (AZI), gentamicin (GEN), rifampin (RIF), and ciprofloxacin, as well as drug combinations such as DOX plus GEN or DOX plus RIF.19,20 However, the recommended treatment of systemic B. henselae infections is based on limited trial data21 with various treatment failures, probably related to antibiotic resistance and bacterial persistence.20,22 Therefore, identifying novel antimicrobials targeting persistent Bartonella pathogens can assist in developing improved therapeutic protocols for the treatment of Bartonella-associated diseases.

Botanical medicines have a long history of documented use and their safety and efficacy are supported by centuries of use in various traditional medicine systems such as Ayurveda and Traditional Chinese Medicine.23 Recent reports have concluded that the frequency of severe adverse effects related to the use of botanical medicine is rare.24–26 Unlike conventional antibiotics, which can have a detrimental impact on the microbiome and increase microbial resistance,27,28 botanical medicines may have a beneficial impact on the microbiome.29 According to the World Health Organization, antibiotic resistance is currently “one of the biggest threats to global health, food security, and development,”30 and therefore, it is important to study botanical medicines with potential antimicrobial activity for potential treatment of antibiotic-resistant bacteria.

Previously, we have developed a rapid high-throughput drug screen method using a SYBR Green I/ propidium iodide (PI) viability assay.31,32 This led to the successful identification of various active drugs or hits as well as drug combinations against stationary phase B. burgdorferi, and this approach has successfully been used as a model of persister drug screens.33–35 In two recent studies, we used this method to screen the Food and Drug Administration (FDA)-approved drug library and a collection of essential oils to identify active candidates with potential for treating Bartonella infections.36,37 In this current study, we utilized the same methodology to screen an herbal product collection which we used recently on Babesia duncani38 and stationary phase B. burgdorferi35 and we identified various botanical extracts with activity against stationary phase B. henselae, which was similarly used as a persister model. The implications of these findings for improved therapy of persistent Bartonella-associated infections are discussed.

Results

Screening the herbal product collection to identify herbs active against non-growing stationary phase B. henselae

Using the SYBR Green I/PI viability assay developed previously,31,36,37 we tested a panel of botanical extracts and their corresponding controls against a 5-day-old stationary phase B. henselae culture in 96-well plates incubated for 3 days. Our goal was to identify those with significant activity against stationary phase B. henselae compared to antibiotic controls. For primary screens, all the herbal products were applied at two concentrations, 1% (v/v) and 0.5% (v/v), and the currently used antibiotics for bartonellosis treatment, such as DOX, AZI, GEN, and RIF, were included as control drugs for comparison. We also included the previously identified FDA-approved drugs effective against B. henselae, including daptomycin (DAP), methylene blue, and miconazole (Table 1).37 All of the pharmaceutical antibiotics were used at their Cmax. In the primary screens, four different alcohol extracts of Juglans nigra, three different alcohol extracts of Cryptolepis sanguinolenta, one alcohol extract of Polygonum cuspidatum, one glycerite extract of P. cuspidatum, one glycerite extract of Scutellaria baicalensis (huang qin), and one glycerite extract of Scutellaria barbata (ban zhi lian) at both 1% (v/v) and 0.5% (v/v) were found to have strong activity against stationary phase B. henselae compared to the control antibiotics AZI, DOX, GEN, and RIF (Table 1). Their concentrations as weight/volume can be calculated based on Table S1, https://links.lww.com/IMD/A8 and are also listed in the Materials and Methods and in Table 1. In contrast, Andrographis paniculate, Stevia rebaudiana, Artemisia annua, Uncaria rhynchophylla, Uncaria tomentosa, Rhizoma coptidis, Citrus paradisi, Dipsacus fullonum, Campsiandra angustifolia, Otoba parvifolia, and colloidal silver did not show significant activity against stationary phase B. henselae (Table S2, https://links.lww.com/IMD/A8). Specifically, the top active herb extracts included J. nigra 30%, 45%, 60%, and 90% alcohol extracts, P. cuspidatum 30% alcohol extract, P. cuspidatum glycerite extract, C. sanguinolenta 30%, 60%, and 90% alcohol extracts, S. baicalensis glycerite extract, and S. barbata glycerite extract. These top hits were identified due to the lower percentage of viable cells remaining compared to the other natural antimicrobials, as well as the fact that they exhibited significantly better activity compared to the current antibiotics used to treat Bartonella infections, including AZI, DOX, GEN, and RIF (P value < 0.05). Because previous experience showed that some compounds in herbal extracts can interfere with the SYBR Green I/PI assay due to autofluorescence, we worked to eliminate this impact by checking the residual cell viability and examining microscope images of the herbal extract-treated samples to confirm the plate reader results. As shown by fluorescence microscopy, solvents such as dimethyl sulfoxide (DMSO) and alcohol did not have significant impact on residual bacterial cell viability compared to the drug-free control (Figure 1A and Table 1) (P value > 0.05). Clinically used antibiotics against Bartonella infections such as AZI and DOX only showed weak activity when used at their Cmax (residual viability above 60%) (Table 1). Antibiotics reported to be clinically active for Bartonella infections, including GEN and RIF showed relatively better activity (residual viability below 50%) against stationary phase B. henselae than AZI and DOX. FDA-approved drugs that we identified as effective against stationary phase B. henselae (DAP, methylene blue, and miconazole)37 had better activity (residual viability below 40%) compared to other antibiotics tested (Figure 1A and Table 1). Among the five top herbal hits, the most active herbal medicines were J. nigra and P. cuspidatum with residual viability between 0% and 16% (Table 1). However, fluorescence microscopy observation of J. nigra 30% and 45% alcohol extracts, and P. cuspidatum glycerite extract at 2.5 mg/mL treatment exhibited a significantly higher percentage of green (viable) cells compared with the plate reader results (Figure 1B and 1C and Table 1) (P value < 0.05). These were also higher than that of most control antibiotics, indicating the relatively poor accuracy of the plate reader results and poor activity of these herbal products at these particular concentrations. Therefore, these were excluded from active hits for subsequent minimum inhibitory concentration (MIC) testing and drug exposure assays (see MIC determination of active hits). Alcohol extracts of different concentrations of C. sanguinolenta also exhibited strong activity against stationary phase B. henselae as shown by red (dead) cells in fluorescence microscope observations (Figure 1B and 1C), which is consistent with the plate reader results. Glycerite extracts from the two Scutellaria plants, including S. baicalensis and S. barbata, also showed good activity with low percentages of residual viable bacterial cells remaining (Figure 1B and 1C).

Table 1 - Activity of top active herbal products against stationary phase Bartonella henselae
HP and control drugs Trade names of HP Residual viability (%) after 1% HP or antibiotic treatment Residual viability (%) after 0.5% HP treatment
Plate reader Microscope Plate reader Microscope
Drug-free control 70% 75%
DMSO control 60% 70%
30% alcohol 70% 78%
60% alcohol 80% 82%
90% alcohol 65% 63%
AZI 70% 65%
DOX 66% 60%
GEN 44% 50%
RIF 31% 45%
DAP 6% 10%
Methylene Blue 29% 35%
Miconazole 40% 50%
Juglans nigra 45% AE Hu tao ren 0%§ 0% 9% 59%
J. nigra 60% AE Black walnut 0%§ 0% 4% 32%
J. nigra 90% AE Black walnut 0%§ 7% 6% 30%
J. nigra 30% AE Black walnut 2% 2% 6% 63%
Polygonum cuspidatum 30% AE Japanese knotweed 6% 0% 8% 21%
P. cuspidatum GE Hu zhang 8% 18% 13% 70%
Cryptolepis sanguinolenta 30% AE Cryptolepis 8% 10% 14% 42%
Scutellaria baicalensis GE Huang qin 8% 12% 11% 25%
Scutellaria barbata GE Ban zhi lian 8% 50% 2% 48%
C. sanguinolenta 60% AE Cryptolepis 10% 24% 14% 34%
C. sanguinolenta 90% AE Cryptolepis 11% 25% 14% 47%
AE: alcohol extract; AZI: azithromycin; DAP: daptomycin; DMSO: dimethyl sulfoxide; DOX: doxycycline; GE: glycerite extract; GEN: gentamicin; HP: herbal products; RIF: rifampin.
The concentrations of herbal products used here can be calculated based on Table S1 and are as follows: 5 mg/mL (1%) or 2.5 mg/mL (0.5%) J. nigra, 5 mg/mL (1%) or 2.5 mg/mL (0.5%) P. cuspidatum, 3.33 mg/mL (1%) or 1.67 mg/mL (0.5%) C. sanguinolenta , 3.33 mg/mL (1%) or 1.67 mg/mL (0.5%) S. baicalensis, 3.33 mg/mL (1%) or 1.67 mg/mL (0.5%) S. barbata. Drug concentrations used in this experiment were based on their Cmax and were as follows: 0.2 μg/mL AZI, 2.4 μg/mL DOX, 10 μg/mL GEN, 7.8 μg/mL RIF, 60 μg/mL DAP, 2.9 μg/mL methylene blue, and 6.3 μg/mL miconazole.
Residual viability was calculated according to the regression equation and the ratio of Green/Red fluorescence obtained by SYBR Green I/PI assay.
Residual viability was assayed by fluorescence microscope counting.
§Values of SYBR Green I/PI calculated by the plate reader were lower than 0% viability.

Figure 1
Figure 1:
Effect of top hit herbal products against stationary phase Bartonella henselae in comparison with control drugs. A 5-day-old stationary phase B. henselae culture was treated with solvent controls or control antibiotics (A), or 1% (v/v) (B) or 0.5% (v/v) (C) herbal products for three days followed by SYBR Green I/PI viability assay and fluorescence microscopy (400× magnification). Drug concentrations used were based on their Cmax and are as follows: 0.2 μg/mL AZI, 2.4 μg/mL DOX, 10 μg/mL GEN, 7.8 μg/mL RIF, 60 μg/mL DAP, 2.9 μg/mL methylene blue, and 6.3 μg/mL miconazole. The concentrations of herbal products used here can be calculated based on Table S1 and are as follows: 5 mg/mL (1%) or 2.5 mg/mL (0.5%) Juglans nigra, 5 mg/mL (1%) or 2.5 mg/mL (0.5%) Polygonum cuspidatum, 3.33 mg/mL (1%) or 1.67 mg/mL (0.5%) Cryptolepis sanguinolenta, 3.33 mg/mL (1%) or 1.67 mg/mL (0.5%) Scutellaria baicalensis, 3.33 mg/mL (1%) or 1.67 mg/mL (0.5%) Scutellaria barbata. Green cells represent live cells and red cells represent dead cells. AE: alcohol extract; AZI: azithromycin; DAP: daptomycin; DMSO: dimethyl sulfoxide; DOX: doxycycline; GE: glycerite extract; GEN: gentamicin; RIF: rifampin.

Time-kill curves of active hits

To further demonstrate the efficacy of the active herbal products identified from the primary screens in eradicating persistent B. henselae cells, we performed a time-kill drug exposure assay against a 5-day-old stationary phase B. henselae culture at a lower concentration of 0.25% (v/v) (at 0.83 or 1.25 mg/mL for the active hits, see Table 2 footnote), along with their corresponding solvent controls. The concentrations of herbal products as weight/volume used in this experiment can be calculated based on Table S1, https://links.lww.com/IMD/A8 and Table 2. The common anti-Bartonella antibiotics, including AZI, DOX, GEN, and RIF, were used at their Cmax as controls. Compared to the drug-free control, as shown in Figure 2A and Table 2, some of the antibiotics such as AZI and DOX showed poor activity in killing stationary phase B. henselae, partly due to their low Cmax. Other antibiotics such as GEN and RIF exhibited better activity and eradicated all B. henselae cells by day 7 and day 5, respectively, to an undetectable level. The difference of residual viabilities of stationary phase B. henselae after treatment by control solvents and without drug treatment was not statistically significant (P value > 0.05). As shown in Figure 2B, all three C. sanguinolenta alcohol extracts of different concentrations were able to eradicate all B. henselae cells to an undetectable level in 7-day drug exposure, where C. sanguinolenta 60% alcohol extract was the most active herbal product that killed B. henselae with no detectable colony forming units (CFUs) after 5-day exposure. J. nigra 60% and 90% alcohol extracts exhibited good activity and eradicated all B. henselae cells without viable cells being recovered after 7-day drug exposure. P. cuspidatum 30% alcohol extract was also effective in killing all B. henselae cells by day 7. However, S. barbata and S. baicalensis showed poor activity at the concentration of 0.83 mg/mL during 7-day drug exposure, with considerable numbers of viable cells remaining after treatment.

Table 2 - Drug exposure assay of top active herbal products against Bartonella henselae stationary phase culture
Herbal products and control drugs CFU/mL after drug exposure
1 Day 3 Days 5 Days 7 Days
Drug-free control 1.45 ± 0.26 × 107 9.17 ± 1.44 × 106 1.67 ± 0.29 × 106 1.33 ± 0.29 × 104
DMSO 2.07 ± 0.12 × 107 9.33 ± 0.58 × 106 1.50 ± 0.50 × 106 1.50 ± 0.50 × 104
30% alcohol 4,17 ± 1.04 × 107 9.50 ± 1.32 × 106 1.33 ± 0.58 × 106 1.83 ± 0.76 × 104
60% alcohol 3.33 ± 0.29 × 107 1.02 ± 0.13 × 107 2.50 ± 1.00 × 106 2.17 ± 0.29 × 104
90% alcohol 3.17 ± 0.29 × 107 9.00 ± 1.50 × 106 1.17 ± 0.29 × 106 4.33 ± 1.15 × 104
AZI 1.87 ± 0.32 × 107 7.00 ± 1.00 × 106 8.83 ± 1.44 × 105 2.17 ± 0.29 × 104
DOX 2.50 ± 1.00 × 107 6.17 ± 1.76 × 106 9.67 ± 2.47 × 105 1.83 ± 1.15 × 104
GEN 5.00 ± 0.00 × 104 1.00 ± 0.00 × 103 8.50 ± 0.87 × 102 0
RIF 5.83 ± 1.76 × 106 5.83 ± 2.57 × 104 0 0
Juglans nigra 60% AE (1.25 mg/mL) 3.67 ± 0.76 × 106 6.67 ± 2.89 × 104 1.02 ± 0.21 × 105 0
J. nigra 90% AE (1.25 mg/mL) 3.83 ± 3.69 × 106 2.67 ± 0.58 × 106 4.83 ± 1.04 × 102 0
C. sanguinolenta, 30% AE (0.83 mg/mL) 6.00 ± 0.87 × 106 2.83 ± 0.76 × 106 6.33 ± 1.26 × 104 0
Cryptolepis sanguinolenta, 60% AE (0.83 mg/mL) 5.33 ± 2.25 × 104 2.67 ± 1.04 × 105 0 0
C. sanguinolenta, 90% AE (0.83 mg/mL) 7.83 ± 2.75 × 106 9.50 ± 3.97 × 105 2.17 ± 1.04 × 102 0
Polygonum cuspidatum 30% AE (1.25 mg/mL) 7.17 ± 1.61 × 106 2.33 ± 0.29 × 106 5.50 ± 3.12 × 102 0
Scutellaria barbata GE (0.83 mg/mL) 1.03 ± 0.20 × 107 3.17 ± 1.04 × 106 3.17 ± 0.58 × 105 3.17 ± 0.58 × 103
Scutellaria baicalensis GE (0.83 mg/mL) 9.00 ± 0.50 × 106 2.83 ± 0.76 × 106 6.50 ± 2.78 × 105 7.83 ± 0.29 × 103
AE: alcohol extract; AZI: azithromycin; CFU: colony forming unit; DMSO: dimethyl sulfoxide; DOX: doxycycline; GE: glycerite extract; GEN: gentamicin; RIF: rifampin.
A 5-day-old stationary phase B. henselae culture was treated with herbal products or control drugs. The beginning CFU for the 5-day-old stationary phase B. henselae culture was about 2 × 107 CFU/mL. At different time points of drug exposure (day 1, day 3, day 5, and day 7), portions of bacteria were removed, washed, and plated on Columbia blood agar for colony forming unit counts.
The herbal product concentration used in this experiment was 0.25% (v/v). The weight concentrations of herbal products used at 0.25% (v/v) can be calculated based on Table S1 and are as follows: 1.25 mg/mL J. nigra, 1.25 mg/mL P. cuspidatum, 0.83 mg/mL C. sanguinolenta, 0.83 mg/mL S. baicalensis, 0.83 mg/mL S. barbata. Drug concentrations used in this experiment were based on their Cmax and were as follows: 0.2 μg/mL AZI, 2.4 μg/mL DOX, 10 μg/mL GEN, and 7.8 μg/mL RIF.

Figure 2
Figure 2:
Time-kill curves of active herbal product treatment against stationary phase Bartonella henselae in comparison with control drugs. The control antibiotics (A) or herbal products (B) were added to the 5-day-old stationary phase culture respectively at time point 0, and at different times of drug exposure (day 1, day 3, day 5, and day 7), portions of bacteria were removed and washed and plated on Columbia blood agar plates for colony forming unit counts. The herbal product concentration used in this experiment was 0.25% (v/v). The concentrations of herbal products used here can be calculated based on Table S1 and are as follows: 1.25 mg/mL Juglans nigra, 1.25 mg/mL Polygonum cuspidatum, 0.83 mg/mL Cryptolepis sanguinolenta, 0.83 mg/mL Scutellaria baicalensis, 0.83 mg/mL Scutellaria barbata. Drug concentrations used in this experiment were based on their Cmax and are as follows: 0.2 μg/mL AZI, 2.4 μg/mL DOX, 10 μg/mL GEN, and 7.8 μg/mL RIF. The dashed lines indicate the limit of detection. AE: alcohol extract; AZI: azithromycin; DAP: daptomycin; DMSO: dimethyl sulfoxide; DOX: doxycycline; GE: glycerite extract; GEN: gentamicin; RIF: rifampin.

MIC determination of active hits

The activity of antibiotics against non-growing bacteria does not always correlate with activity against growing bacteria.37 Thus, it was also important to determine the MICs of these active herbal medicines against log phase growing B. henselae. The MIC determination of herbal medicines for B. henselae was conducted by the standard microdilution method as described.36,37 As shown in Table 3, J. nigra 60% extract was the most active herbal product among the top 5 hits, capable of inhibiting visible B. henselae proliferation at 0.625–1.25 mg/mL. Other herbal medicines, including C. sanguinolenta 30%, 60%, and 90% alcohol extracts, S. baicalensis, and S. barbata had similar activity against growing B. henselae and inhibited log phase B. henselae proliferation at 0.83–1.67 mg/mL. J. nigra 90% alcohol extract and P. cuspidatum 30% alcohol extract inhibited log phase B. henselae proliferation at 1.25–2.5 mg/mL. These results indicated that these herbal medicines were active against non-growing stationary phase B. henselae, and also effective against log phase growing B. henselae.

Table 3 - MICs of top active herbal products against Bartonella henselae
Herbal Products or Antibiotics MIC (μg/mL)
Juglans nigra 60% AE 625–1250
Cryptolepis sanguinolenta 30% AE 830–1670
C. sanguinolenta 60% AE 830–1670
C. sanguinolenta 90% AE 830–1670
S. baicalensis GE 830–1670
S. barbata GE 830–1670
J. nigra 90% AE 1250–2500
Polygonum cuspidatum 30% AE 1250–2500
Rifampin <0.01
Azithromycin 0.04–0.08
Doxycycline 0.08–0.16
Gentamicin 0.63–1.25
AE: alcohol extract; GE: glycerite extract; MICs: minimum inhibitory concentrations.

Discussion

In this study, we successfully applied the SYBR Green I/PI viability assay to evaluate a panel of botanical medicines for their activity against stationary phase B. henselae as a model of persister drug screens.36,37 We identified herbal medicines with high activity at 3.33 mg/mL or 5 mg/mL concentrations compared with clinically used antibiotics, including extracts of J. nigra, C. sanguinolenta, P. cuspidatum, S. baicalensis, and S. barbata. Among these top hits, three herbal product extracts were able to eradicate all stationary B. henselae cells, with no CFUs being detected after 7-day drug exposure at a low concentration of 0.83 mg/mL or 1.25 mg/mL. These included C. sanguinolenta 30%, 60%, 90% alcohol extracts, J. nigra 60%, 90% alcohol extracts, and P. cuspidatum 30% alcohol extracts. The MIC determination of these active hits showed they were also effective in inhibiting the growth of log phase B. henselae.

The herbal medicine extracts active against B. henselae have also been reported to have various biological activities in previous research. Studies on different components and species of the genus Juglans have shown pain-relieving, antioxidant, antibacterial, antifungal, and antitumor activities.39 In particular, J. nigra exhibited both bacteriostatic activity and bactericidal activity against Borrelia based on in vitro studies.35Juglans spp. contain several active constituents with potential importance to human health, including juglone, phenolic acids, flavonoids, and catechins (including epigallocatechin).40 The safety of J. nigra use in humans has not been adequately studied; however, it has a long history of anecdotal use and the oral LD50 of juglone in rats has been calculated at 112 mg/kg.41

C. sanguinolenta and its constituents have been reported to have antibacterial, antifungal, antiparasitic, anti-inflammatory, and anticancer properties.42 A recent review has assessed the phytochemistry and pharmacology of C. sanguinolenta, and concluded that although there may be some concern regarding potential reproductive toxicity, it is generally safe at doses below 500 mg/kg and may serve as a promising source of potential antimicrobial agent(s).42 Among constituents and secondary metabolites of the plant, an alkaloid called cryptolepine is the most well-studied. Cryptolepine was reported to have a lytic effect on Staphylococcus aureus as seen in scanning electron microscopy graphs which led to altered cell morphology and was able to intercalate into DNA at cytosine-cytosine sites and inhibited the activity of topoisomerase, causing DNA damage.43–45

P. cuspidatum and its constituents have been shown to have antimicrobial, anti-tumor, anti-inflammatory, neuroprotective, and cardioprotective effects.46–49 One of the most active constituents is a polyphenol called resveratrol, which was reported to be active against log phase B. burgdorferi and Borrelia garinii by in vitro testing.50 In addition, another active constituent of P. cuspidatum called emodin (6-methyl-1,3,8-trihydroxyanthraquinone) has been shown to have activity against stationary phase B. burgdorferi cells.51 A study investigating the mechanism of action of P. cuspidatum using a network pharmacology approach suggested that the component polydatin might play a pivotal role in the therapeutic effects of P. cuspidatum.52 Recent trials using P. cuspidatum have not reported significant toxicity.53,54

In this current study, clinically used antibiotics for treating Bartonella-associated infections, including AZI and DOX, showed weak activity in eradicating stationary phase B. henselae cells (Table 1, Figure 1, and Figure 2). This finding coincides with the reported discrepancies in antibiotic efficacies between in vitro MIC data and clinical data from patients.19 The poor activities of current clinically used antibiotics against stationary phase B. henselae as shown in our study can partly explain clinically documented treatment failure due to suspected persistent infections. This phenomenon may also be partly due to the limited antibacterial activity of these antibiotics. These antibiotics all target growing bacteria and they are not very effective at killing non-growing stationary phase B. henselae, and thus can lead to treatment failure due to persistent and chronic infections. Our screening method using the SYBR Green/PI assay detects dead cells based on PI influx through a damaged cell membrane. Therefore, our assay will exclude any bacteriostatic antimicrobial activity that does not alter the bacterial membrane permeability in non/slow-growing cells, and thus helps to identify drugs that have cidal activity against non-growing cells. Since activity against non-growing bacteria has been shown to be important for eradication of persistent infections,55 the herbal extracts we identified that have activity against both growing and non-growing B. henselae can be promising candidates for treating persistent Bartonella infections, as they contain multiple active phytochemicals, including flavones and tannins. These compounds have complex and synergistic effects and thus have potentially broader antimicrobial activity. Many of these phytochemicals are lipophilic and can target the bacterial cell membrane, an important target of persister drugs like pyrazinamide56 and DAP.31 The high lipophilicity of these phytochemicals can induce bacterial cell membrane damage,43 and may explain occurrence of aggregated bacterial forms seen in microscopic pictures (Figure 1).

Although additional studies will be needed to clarify the extent to which our current in vitro data can be translated into clinical practice, it should be noted that all three of the most promising botanicals identified (C. sanguinolenta, J. nigra, and P. cuspidatum) do have existing data indicating potential systemic effects in vivo. While only a limited number of human studies have been conducted to date using P. cuspidatum as a whole herb, the available data show a significant reduction in inflammation and oxidative stress following oral administration.54,57P. cuspidatum contains over 60 active constituents58 with varying degrees of bioavailability and wide-ranging mechanisms of action.52 The most well studied of these active constituents is resveratrol, which has been shown in epidemiology studies to prevent cardiovascular disease59 and certain cancers,60,61 and clinical trials have shown significant benefits in inflammation,62 hypertension,63 hypertriglyceridemia,64 hypercholesterolemia,65 endothelial cell function,66 metabolic syndrome,67 weight loss,68 and cognitive performance.69C. sanguinolenta may be uniquely positioned to treat hemotropic infections such as Bartonella based on the two preliminary human trials showing efficacy in treating acute uncomplicated malaria.70 The most well-studied active constituent in C. sanguinolenta is cryptolepine, which exhibits low bio-availability, moderate plasma half-life (4.5 hours), and extensive distribution.71 The pharmacokinetics may be improved by using a nanoformulation of cryptolepine, which exhibits a stronger anti-plasmodial effect, improved bioavailability, and longer half-life compared to unaltered cryptolepine.72 Clinical studies are lacking on J. nigra as a whole herb; however, data from two active constituents indicate a systemic effects in humans. Juglone, an active constituent in J. nigra, exhibits a bioavailability of 40%–50%73 while a liposomal formulation of juglone exhibits a significantly longer half-life compared to unaltered juglone.74 The most well-studied active constituent in J. nigra is epigallocatechin gallate (EGCG), which has been documented to have a favorable pharmacokinetic profile in humans,75 with human studies revealing significant benefits for multiple diseases, including hyperlipidemia,76 metabolic syndrome,77 acne,78 breast cancer,79 and upper respiratory infections.80 Of interest, both bartonellosis and coronavirus disease 2019 (COVID-19) can induce hemolysis, endotheliopathy, and heme related dysfunctions11,81–83 and C. sanguinolenta, resveratrol and EGCG have all been proposed as potential therapeutic agents for COVID-19.84–86

Conclusion

This current study is the first to identify the in vitro antimicrobial activity of extracts from J. nigra, C. sanguinolenta, and P. cuspidatum against stationary phase B. henselae. Given the possibility of B. henselae coinfection among Lyme and tick-borne disease patients and the overlapping activity of C. sanguinolenta, and P. cuspidatum against B. henselae, B. burgdorferi,35 and B. duncani,38 and J. nigra against B. henselae and B. burgdorferi,35 these botanical medicines offer promising potential therapeutic agents for improving treatment outcomes for co-infections caused by these organisms. Future studies are needed to identify the active ingredients and to better understand specific antimicrobial mechanisms of action. It would also be of interest to test the active components juglone, cryptolepine, resveratrol, EGCG, and emodin on B. henselae in future studies. Furthermore, different parts of these plants can have varied antimicrobial activity and pharmacokinetic profiles, and different solvents used to extract the compounds can also significantly affect their activity.

Since the present study only tested the activity of herbal medicines against B. henselae in vitro, there are a few additional points that are important to address. For one, B. henselae is a facultative intracellular pathogen and can reside and propagate inside mammalian erythrocytes and/or endothelial cells. Therefore, in the future, assessing the activity of these identified candidates against intracellular B. henselae ex vivo and in vivo in animal models of infection87 will be important. The host cell can provide the pathogen protective shelter from the activity of pharmaceutical and/or herbal antimicrobials as well as from the host immune system; therefore, the efficacy of these antimicrobials in vivo might differ from that in vitro.11 Additionally, it is important to note that botanical medicines have multiple mechanisms of action beyond just the antimicrobial activity assessed in the present study. For example, botanicals have been shown to exert effects via multiple mechanisms with potential benefit for Bartonella infections, including anti-inflammatory activity, immune modulation/stimulation, microbiome modulation, endothelial cell support, and biofilm disruption. Future studies are needed to assess the safety and efficacy of these herbal medicines in appropriate animal models of Bartonella infections where broader biologic mechanisms of action can be evaluated.

In the future, we also hope to test different combinations of active herbal medicines and their active constituents with and without antibiotics to develop better treatment combinations. As indicated by the Yin-Yang model, bacterial pathogens have heterogeneous subpopulations, with persister populations (Yin) and growing populations (Yang), with various subpopulations having varying metabolic or dormant states in continuum.22 Therefore, drug/herb or herb/herb combinations can provide more effective treatments, targeting diverse bacterial subpopulations and physiological states. Indeed, members of our group recently demonstrated that antibiotic combinations were more effective in eradicating in vitro stationary phase and biofilm B. henselae compared to single antibiotics.88 Our goal is to use the herbal medicines we identified in this study to develop novel safe and effective treatments for persistent bartonellosis.

Materials and methods

Bacterial strain, culture media, and culture conditions

B. henselae JK53 strain (obtained from BEI Resources/ATCC, NIAID, NIH) was cultured in Schneider's Drosophila medium (Life Technologies Limited, Paisley, UK) supplemented with 10% fetal bovine serum (Sigma-Aldrich, Co., St. Louis, MO, USA) and 5% sucrose (Fisher Scientific, New Jersey, USA) as described previously.37,89 Cultures were incubated in sterile 15 mL or 50 mL polypropylene conical tubes (Corning, New York, USA) in a microaerophilic incubator without shaking at 37 °C, 5% CO2. Based on our previous study, the 1-day-old and 5-day-old cultures were considered as log phase and stationary phase, respectively.37 Columbia sheep blood agar (HemoStat Laboratories, Dixon, CA, USA) was used to perform the colony counts in drug exposure assays, and was also cultured at 37 °C, 5% CO2.

Drugs and botanical medicines

A panel of botanical medicines was purchased from KW Botanicals Inc. (San Anselmo, CA, USA), Heron Botanicals (Kingston, WA, USA), and Hawaii Pharm LLC (Honolulu, HI, USA) as described previously.35 The botanical medicines were chosen based on significant antimicrobial activity against other bacterial pathogens shown by previous studies,34,37,50,90 anecdotal clinical usage reported by clinicians and patients, favorable safety profiles, and the ability to be absorbed systemically. Most of the botanicals were identified via macroscopic and organoleptic methods with voucher specimens on file with the respective production facilities. Additional details on sourcing, testing, validation, and concentrations of botanical and natural medicines used are summarized in Table S1, https://links.lww.com/IMD/A8. Most botanical extracts were provided as alcohol extracts in 30%, 60%, and 90% alcohol dilutions, and the alcohol used was also tested separately as a control in the same dilutions of 30%, 60%, and 90%. Herbal products were dissolved in DMSO at 5% (v/v), followed by dilution at 1:5 into 5-day-old stationary phase cultures to achieve 1% final concentration. To make further dilutions for evaluating anti-Bartonella activity, the 1% herbal products were further diluted with the same stationary phase cultures to achieve desired concentrations. The percentage and weight/volume conversions for the active hits are as follows: 1% or 0.5% of J. nigra and P. cuspidatum extracts are equivalent to 5 mg/mL or 2.5 mg/mL respectively, while 3.33 mg/mL (1%) or 1.67 mg/mL (0.5%) for C. sanguinolenta, S. baicalensis, and S. barbata. Antibiotics, including AZI, DAP, DOX, GEN, methylene blue, miconazole, and RIF included as controls, were purchased from Sigma-Aldrich (USA) and dissolved in appropriate solvents88 to form 10 mg/mL stock solutions. All the antibiotic stocks were filter-sterilized by 0.2 μm filters except the DMSO stocks and then diluted and stored at −20 °C.

Microscopy techniques

B. henselae cultures treated with different herbal medicines or control drugs were stained with SYBR Green I (10× stock, Invitrogen, Waltham, MA, USA) and PI (60 μM, Sigma) mixture dye and then examined with a BZ-X710 All-in-One fluorescence microscope (KEYENCE, Inc., Osaka, Japan). The SYBR Green I/PI pre-mixed dye was added to the sample in a 1:10 ratio of the dye to sample volume and mixed thoroughly, followed by incubating in the dark at room temperature for 15 minutes. SYBR Green I is a green permeant dye that stains all cells whereas PI is an orange-red impermeant dye that only stains dead or damaged cells with a compromised cell membrane. Thus, live cells with an intact membrane will be stained green by SYBR Green I, while damaged or dead cells with a compromised cell membrane will be stained orange-red by PI. The residual bacterial viability was then assessed by calculating the ratio of green/red fluorescence as described previously.32 The stained samples were confirmed by analyzing three representative images of the same bacterial cell suspension using fluorescence microscopy. BZ-X Analyzer and Image Pro-Plus software were used to quantitatively determine the fluorescence intensity.

Screening of herbal product collection against stationary phase B. henselae using the SYBR Green I/PI viability assay

For the primary screening, each product was assayed in two concentrations, 1% (v/v) and 0.5% (v/v). A 5-day-old stationary phase B. henselae culture was used for the primary screen. First, 40 μL 5% herbal product DMSO stocks were added to 96-well plates containing 160 μL B. henselae culture to obtain the desired concentration of 1%. Then the 0.5% concentration was obtained by mixing 100 μL 1% treatment with 100 μL B. henselae culture. Antibiotics (AZI, DAP, DOX, GEN, methylene blue, miconazole, and RIF) were used as control drugs at their Cmax, a pharmacokinetic measure referring to the maximum serum concentration that a drug achieves in a specified compartment (such as blood) after administration. Control solvents (DMSO, 30%, 60%, and 90% alcohol) were also included. Plates were sealed and placed in a 37 °C incubator without shaking over for three days. As previously described,32 to assess viability and measure live/dead cell ratios after drug exposure, the SYBR Green I/PI dye was added to the sample followed by incubation in the dark for 15 minutes. Then the plate was read by microplate reader (HTS 7000 plus Bioassay Reader, PerkinElmer Inc., Waltham, MA, USA). The green/red (538 nm/650 nm) fluorescence ratio of each well was used for calculating the residual viability percentage, according to the regression equation of the relationship between residual viability percentage and green/red fluorescence ratio obtained by least-square fitting analysis.37 All tests were run in triplicate.

Drug exposure assays of active hits

The active hits from the primary screens were further confirmed by drug exposure assays. The 5-day-old stationary phase B. henselae cultures were treated with 0.25% (v/v) active herbal medicines. Control antibiotics (AZI, DAP, DOX, GEN, methylene blue, miconazole, and RIF) were again used at their Cmax. Control solvents were also included. The drug exposure assay was carried out in 15 mL polypropylene conical tubes over the course of 7 days at 37 °C, 5% CO2 without shaking. At each time point, a portion of bacterial cells was collected by centrifugation and rinsed twice with fresh Schneider's medium followed by resuspension in fresh Schneider medium. Then the cell suspension was serially diluted and plated on Columbia blood agar plates for viable bacterial counts (CFU). The plates were incubated at 37 °C, 5% CO2 until visible colonies appeared and the CFU/mL was calculated accordingly. All tests were run in triplicate.

MIC determination of active hits

The standard microdilution method was used to measure the MIC of each herbal product needed to inhibit the visible growth of B. henselae after a 5-day incubation period as described.36,37 A diluted 1-day-old B. henselae log phase culture was used for MIC determination. The 5% herbal product stocks were added into 96-well plates containing 1 × 106 bacteria per well with fresh modified Schneider medium to achieve 1% final concentration. Other lower concentrations were obtained by mixing 1% treatment with diluted 1-day-old log phase B. henselae cultures. Plates were sealed and incubated at 37 °C without shaking for five days. Then the bacterial cell proliferation was assessed using the SYBR Green I/PI assay and the bacterial counting chamber. The MIC is the lowest concentration of the herbal product that prevented the visible growth of B. henselae. All tests were run in triplicate.

Statistical analysis

The statistical analysis was performed using two-tailed Student t-test. Mean differences were considered statistically significant if P < 0.05. Analyses were performed using Image Pro-Plus, GraphPad Prism, and Microsoft Office Excel.

Acknowledgments

The authors thank herbalists Eric Yarnell ND and Brian Kie Weissbuch for providing botanical medicine extracts for evaluation in this study. The authors thank BEI Resources/ATCC for providing the B. henselae JK53 strain used in this study. The authors gratefully acknowledge the support of their work by the Bay Area Lyme Foundation, the Steven & Alexandra Cohen Foundation, LivLyme Foundation, and the Einstein-Sim Family Charitable Fund.

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Keywords:

antimicrobial activity; Bartonella henselae; botanical medicine; herbal medicine; medicinal plant; persisters; phytotherapy; stationary phase

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